PROJECT SUMMARY Children with a genetic disease or birth defect are hospitalized at a younger age, stay longer, and have a higher death rate than children hospitalized for other reasons. One in 33 infants born in the US has a birth defect; the number one cause of infant mortality. Our long-term goal is to change these dire statistics by delineating the mechanisms that reduce game quality by increasing mutagenesis in male gametes with increasing age, i.e., the paternal age effect. The paternal age effect is increasingly significant with increasing birth rates to older fathers, and is directly relevant to male reproductive health and child health. Previously published studies revealed that reduced base excision repair protein APE1, results in reduced base excision repair and increased mutagenesis in spermatogenic cells with increasing age. Preliminary data leads us to test the hypothesis that MDM2 ubiquitination of APE1 is triggered by TRP53 Ser18/23 phosphorylation resulting in reduced APE1. Aim 1: Test whether MDM2 ubiquitinates APE1, leading to greater amounts of highly ubiquitinated APE1 in germ cells of older mice, proteasomal degradation of APE1, and a greater spontaneous mutation frequency. Aim 2: Test whether phosphorylation of TRP53 at Ser18/23 triggers degradation of APE1 in spermatogenic cells from older mice. Aim 3: Test whether changes in APE1 abundance, and mechanisms driving those changes, originate in the spermatogonial stem cell population. Methods: Defined spermatogenic cells will be prepared from male mice carrying targeted changes in Mdm2 and Trp53 to test whether these tumor suppressors regulate APE1 abundance in young mice and become chronically activated in old wild type mice resulting in reduced APE1 abundance, reduced base excision repair and increased mutagenesis. The importance of proteasome degradation and MDM2 activity will be tested using inhibitors. CometChip arrays will analyze DNA damage in single cells to determine if increased DNA damage may trigger TRP53 activation. Duplex tag next generation sequencing will determine if increases in mutation frequency initiate in the spermatogonial stem cell population. We propose a novel model in which tumor suppressors that function normally to safeguard genome integrity, instead cause decreased gamete quality and increased mutagenesis by reducing APE1 abundance in the unique biological context of germ cells and aging. This is a paradigm shift from the widely-held view of mutations accumulating passively in stem cells as the major driving force of reduced genetic quality in male gametes with aging and for the role of TRP53 and MDM2 as protectors of the genome.